Delivery and activation through liposome incorporation of diaminocyclohexane platinum(II) complexes

ABSTRACT

Antitumor compositions are disclosed, as well as methods of preparing the compositions and methods of using them to inhibit tumor growth in mammals. The invention can take advantage of intraliposomal conversion of a platinum complex having the formula                    
     where R 1  is diaminocycloalkyl, and R 2  and R 3  each have the formula                    
     where R 4 , R 5 , and R 6  are each independently hydrocarbon moieties having from 1 to about 10 carbon atoms, 
     into a complex having the formula 
     
       
         R 1 —Pt—X 2   (II)  
       
     
     where X is halogen.

This application is a division of U.S. Ser. No. 08/764,095 filed on Dec.6, 1996 now U.S. Pat. No. 5,843,475.

BACKGROUND OF THE INVENTION

The present invention relates to methods and compositions for thetreatment of cancer.

One drug that has proven effective in the treatment of certain tumors iscisplatin (cis-dichlorodiamine-platinum(II)). However, cisplatin hascertain disadvantages. For example, its use in some circumstances islimited by its toxicity to the patient, especially its nephrotoxicity.As another example, tumors sometimes develop resistance to cisplatin.

In an effort to overcome the disadvantages of cisplatin, researchershave synthesized and tested various other platinum complexes aspotential antitumor agents. One such compound isdichloro(1,2-diaminocyclohexane) platinum(II) (referred to in theremainder of this patent as “DACH—Pt—Cl₂”). However, this compound hasvery low solubility in water, making it impractical for formulation andadministration in aqueous solution. Further, although various platinumcomplexes have been formulated in liposomes in the past, a liposomalformulation of DACH—Pt—Cl₂ has not been developed because that complexis insoluble in most organic solvents. Although it has good solubilityin dimethylformamide, that solvent has a very high boiling point,therefore making it impossible or impractical to prepare a liposomalformulation of DACH—Pt—Cl₂ using standard evaporation methods.

Other platinum-based antitumor drugs, such ascis-bis-neodecanoato-trans-R,R-1,2-diaminocyclohexane platinum (II)(NDDP) have been prepared and tested as antitumor agents. However, aneed still exists for improved antitumor drug formulations that havegood antitumor activity, low toxicity to non-cancerous cells in apatient, and desirable storage characteristics.

SUMMARY OF THE INVENTION

The present invention relates to a liposomal antitumor composition andto methods of using the composition to inhibit tumor growth in mammals.The invention also concerns methods of preparing the antitumorcomposition.

The present invention can take advantage of intraliposomal conversion ofa platinum complex having the formula

into a complex having the formula

R₁—Pt—X₂  (II)

where X is halogen. This makes it possible to prepare liposomalformulations of the complex (II), which had not been practicalpreviously due to its low solubility in water and most organic solvents.

In the above complexes, R₁ is diaminocycloalkyl, preferably a1,2-diaminocycloalkyl group having from about 3 to about 6 carbon atoms,most preferably 1,2-diaminocyclohexane. R₂ and R₃ each have the formula

where R₄, R₅, and R6 are each independently hydrocarbon moieties havingfrom 1 to about 10 carbon atoms, preferably alkyl having from 1 to about6 carbon atoms, most preferably alkyl having from 1 to about 3 carbonatoms. R₂ and R₃ can be the same but do not have to be the same.Likewise, R₄, R₅, and R₆ can be the same but do not have to be the same.X is most preferably chlorine.

One aspect of the present invention is a liposomal antitumor compositionthat comprises the complex (II) entrapped in a liposome. In a particularembodiment of the invention, the liposome comprises an acidicphospholipid, for example dimyristoyl phosphatidyl glycerol. Withoutbeing bound by theory, it is believed that the presence of the acidicphospholipid in the liposome enhances or accelerates the conversion ofthe complex (I) to the complex (II).

Another aspect of the present invention is a method of inhibiting tumorgrowth. The method comprises administering to a mammal a compositionthat comprises the complex (II) entrapped in a liposome, with theplatinum complex being present in an amount effective to inhibit tumorgrowth.

Another aspect of the present invention is a method of preparing theantitumor composition. This method comprises the step of adjusting thepH of a composition that comprises the platinum complex (I) entrapped ina liposome, whereby the pH is made somewhat acidic, preferably betweenabout 2 and about 6.5, resulting in the conversion of the complex (I)into the complex (II). The resulting composition can then beadministered to a patient.

In one particular embodiment of this method, the complex (I) isconverted to the complex (II) within the liposome. This allows aliposomal formulation of the complex (I) to be manufactured and stored,and then shortly before administration to a patient, the liposomalformulation of complex (I) can be converted to a liposomal formulationof complex (II) in situ by simply adding an acidic solution to theformulation. Optionally, after a predetermined time has passed since theaddition of the acidic solution, the pH can be readjusted, preferably toat least about 7, in order to stop the conversion of complex (I) to(II).

A broader aspect of the invention concerns a method of delivering abiologically active chemical moiety internally to a mammal. Thebiologically active moiety can be, for example, an antitumor agent. Themethod comprises (a) providing an aqueous formulation of a prodrug ofthe biologically active moiety, the prodrug being entrapped in aliposome, and the prodrug further being capable of forming thebiologically active moiety upon exposure to a solution having an acidicpH; (b) reducing the pH to an acidic level, thereby converting theprodrug to the biologically active compound; and (c) administering theaqueous formulation to a mammal. Administration of the liposomalformulation to the mammal can suitably be done after the conversion ofthe prodrug, but it might also be done before conversion, such that theconversion would then occur in vivo, for example due to acidiccomponents (e.g., acidic phospholipids) of the liposome.

The present invention has a number of advantages over prior art platinumantitumor formulations and methods, including better antitumor activity,greater potency, and reduced toxicity to non-cancerous cells of thepatient. Further, the compositions of the present invention permit theformulation in liposomes and the delivery of platinum complexes thatcould not be so formulated in the past.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Chemical structure of NDDP, B10, and L10.

FIG. 2. Effect of structure of Pt-complex, DMPG content, and aqueoussolution on the intraliposomal stability and cytotoxicity of NDDP (),B10 (◯), and L10 (▪). FIG. 2A—DMPC:DMPG=7:3, saline; FIG.2B—DMPC:DMPG=7:3, PBS; FIG. 2C—DMPC:DMPG=3:7, saline; FIG.2D—DMPC:DMPG=3:7, PBS. IC₅₀ values are in g/ml.

FIG. 3. Effect of lipid composition on the intraliposomal stability ofNDDP. 0.9% NaCl aqueous solution (starting pH=7.0) was used asreconstituting solution of liposomes and the final pH was checked at 6 hafter liposome preparation.

FIG. 4. ¹⁹⁵Pt NMR of liposomal NDDP suspension. FIG. 4A—¹⁹⁵Pt NMR ofSample 1 in chloroform prepared by extraction with CHCl₃ from liposomalNDDP suspension reconstituted in saline and kept for 6 h at roomtemperature. FIG. 4B—¹⁹⁵Pt NMR of Sample 2 in CH₃OH prepared byreconstitution of liposomal NDDP in saline for 6 h, lyophilization ofwater for 2 d, and redissolution of mixtures in CH₃OH. FIG. 4C—¹⁹⁵Pt NMRof Sample 3 in CH₃OH prepared by evaporation of t-butanol andredissolution of mixtures in CH₃OH.

FIG. 5. FIG. 5A—¹H-¹H correlated spectroscopy of DACH—Pt—Cl₂ in DMF-d₇.FIG. 5B—¹³C NMR of DACH—Pt—Cl₂ in DMF-d₇.

FIG. 6. Sample preparation for NMR tracking experiment of liposomalNDDP. FIG. 6A—with lipids in chloroform solutions. FIG. 6B—with lipidsin dry powder.

DESCRIPTION OF SPECIFIC EMBODIMENTS

Cis-bis-neodecanoato-trans-R,R-1,2-diaminocyclohexane platinum (II)(NDDP) is a lipophilic platinum complex (Pt-complex) that can beformulated in a liposomal carrier. Various details about the making anduse of NDDP and other platinum complexes are disclosed in U.S. Pat. Nos.5,041,581, 5,117,022, 5,186,940, 5,178,876, and 5,384,127. Those patentsare incorporated here by reference.

Prior studies have suggested that NDDP is a pro-drug that exerts itsbiological activity through activation within the liposome bilayers. Inorder to understand the kinetics of the intraliposomaldegradation/activation of different liposomal Pt-complexes, we studiedthe effects that the structure of the Pt-complex, pH, temperature, lipidcomposition, content of acidic phospholipids, liposome size, andpresence of residual chloroform have on the stability, in vitrocytotoxicity, and in vivo antitumor activity of different liposomalPt-complex preparations. The following factors were found to enhance theintraliposomal degradation/activation of Pt-complexes: 1) the size andspatial configuration of the Pt-complex, 2) an acidic pH, 3) a hightemperature, 4) the presence and amount of acidic phospholipids, and 5)the presence of residual chloroform. Liposome size did not affect theintraliposomal stability of different Pt-complexes.

A good inverse relationship between the extent of drug degradation andin vitro cytotoxicity and between the extent of drug degradation and invivo antitumor potency was observed, thus confirming that the biologicalactivity of these complexes is exerted through the intraliposomalformation of certain active intermediate(s). The only activeintermediate that could be identified wascis-bis-dichloro-trans-R,R-1,2-diaminocyclohexane platinum (II) whosestructure was confirmed by ¹H, ¹³C, and ¹⁹⁵Pt nuclear magnetic resonanceEMR) spectroscopy.

We have previously developed liposomal formulations of lipophilicPt-complexes for in vivo administration [5,6] and have studied theirchemical and biological properties. [1,4,7] The general structure of thepreferred Pt-complexes used is [DACH—Pt—R₂], where DACH istrans-R,R-1,2-diaminocyclohexane and R is a lipophilic carboxylatogroup. The Pt-complex is thought to intercalate between the phospholipidmolecules of the lipid bilayers of the liposomes. The most remarkablecharacteristic of these complexes is that they are not cross-resistantwith cisplatin, both in vitro and in vivo. [1,5] The leadingformulation, liposomal cis-bis-neodecanoato-DACH-platinum (II) (NDDP)uses large liposomes composed of dimyristoylphosphatidyl choline (DMPC)and dimyristoylphosphatidyl glycerol (DMPG) at a 7:3 molar ratio and isnow in clinical trials. Interestingly, liposomal-NDDP must undergo achemical degradation/activation process into an active intermediate(s)within the liposomes shortly after liposome preparation in order toexert its antitumor activity. [7] We have previously reported that thischemical reaction depends on the content of DMPG in the lipid bilayer,and based on this finding, we hypothesized that a DMPG-Pt complex mightbe one of the active intermediates. We have also reported that thestructure of the Pt-complex has an effect on the intraliposomal drugstability: the compounds with linear and short carboxylato leavinggroups are more stable and less potent than the compounds with branchedor longer linear leaving groups. [8] A full characterization of theactive intermediate(s) as well as the different factors that influencethe degradation/activation process is mandatory for the development ofone of these agents as a pharmaceutical product.

For that purpose, we selected NDDP (highly branched structure) and twoisomers, B10 (minimally branched structure) and L10 (linear structure)(FIG. 1) and studied the relationship between their biological activityand their intraliposomal stability. We examined the effect of pH,temperature, lipid composition, liposome size, and presence of residualchloroform on the degradation of the Pt-complexes, and attempted toidentify the active intermediate(s) by tracking experiments using ³¹Pand ¹⁹⁵Pt NMR spectra. Our results indicate that thedegradation/activation of these Pt-complexes is greatly dependent on thepH of the suspension, and that DACH-dichloroplatinum (DACH—Pt—Cl₂) isthe only intermediate that can be identified, thus suggesting that thesePt-complexes are prodrugs of DACH—Pt—Cl₂ when incorporated in liposomessuspended in saline.

MATERIALS AND METHODS Preparation of Liposomal Pt-complex

NDDP, B10, and L10 were synthesized as previously described [1,2] andrecrystallized in acetone. DMPC, DMPG, dioleyl phosphatidyl choline(DOPC), dioleyl phosphatidyl glycerol (DOPG), phosphatidic acid (PA),phosphatidyl ethanolamine (PE), and phosphatidyl serine (PS) werepurchased from Avanti Polar Lipids (Alabaster, Ala.).

Multilamellar vesicles containing Pt-complexes were prepared by thelyophilization method using the lipids in chloroform solutions or in drypowder.

Method 1: With chloroform solutions of lipids [1], lipids were mixed atthe desired molar ratio, and the chloroform was removed in a rotaryevaporator. To the dried lipid film, t-butanol solutions of Pt-complexwere added and shaken at 40° C. for 10 min. The solutions were thenfrozen in a dry-ice-acetone bath, and t-butanol was removed bylyophilization overnight to give lyophilized preliposomal powder.

Method 2: With lipids in dry powder, lipids were mixed and dissolved int-butanol:water (10:1/v:v). To this solution, t-butanol solutions ofPt-complex were added, and the rest of the procedure was the same asdescribed above.

Saline or PBS was added (1 ml/mg of Pt-complex) to reconstitute thelyophilized preliposomal powder, and the suspension was hand-shaken for10 min to obtain large-size liposomes. Small-size liposomes wereprepared by sonication of large-size liposomes for 1 min with anultrasonic cell disrupter (Laboratory Supplies Co., New York, N.Y.). Thesize distribution of the different liposomal preparations was determinedwith a Nicomp Submicron Particle Sizer Model 370 (Nicomp Particle SizingSystems, Santa Barbara, Calif.).

Intraliposomal Stability

Stability of the different Pt-complexes incorporated in liposomes wasdetermined as described previously [8] by comparing the HPLC profiles asa function of time. In brief, aliquoted samples of liposome suspensionwere diluted (7×) with methanol at 0, 2, 6, and 24 h after liposomepreparation, and each sample was then monitored by HPLC using chromega-8bond column (4.6 mm×25 cm, 8 μm: ES Industries, N.J.) and 10%water-methanol as eluant. The flow rate was 1 ml/min, and the complexeswere detected by UV at 224 nm wavelength.

Biological Activity

In vitro. The in vitro cytotoxicity of liposomal Pt-complexes againstA2780 human ovarian carcinoma cells was assessed by the MTT dyereduction assay. In brief, A2780 cells were seeded in 96-well plates,allowed to attach overnight, and then exposed to various concentrationsof drugs for 20 h. After washing the cells with PBS, fresh medium wasadded for 52 hours and the cell survival fractions determined by MTTassay.

In vivo. The in vivo antitumor activity of liposomal Pt-complexes wasassessed against intraperitoneal L1210 mouse leukemia. Groups of 6-8mice weighing 18-20 g were inoculated with 106 cells (0.2 ml, i.p.) onday 0, and treatment (25, 50, 100 and 150 mg/kg) was started on day 1(0.15-0.5 ml, i.p.). The results were expressed as the median survivalof treated animals divided by the median survival of control animals×100(% T/C).

Identification of Active Intermediates

To characterize the active intermediate(s) in the reaction cascade ofL-NDDP reconstituted in saline (0.9% NaCl), tracking experiments using¹⁹⁵Pt NMR spectra in combination with ³¹P NMR were performed. Theprocedures for the preparation of the samples is summarized in FIG. 6.Samples 1-3 were prepared using the lipids DMPC and DMPG purchased inchloroform solution, while samples 4-8 were prepared using lipids in drypowder. In samples 1-3, the chloroform was initially evaporated in arotavapor. The lipid film was dissolved in t-butanol containing the NDDPin solution. An aliquot of this solution was kept at 40° C. for 6 h andthen lyophilized and extracted with methanol (sample 3). The remainingwas lyophilized immediately, thus resulting in a preliposomal powder,which was reconstituted with saline to produce the liposome suspension.The liposome suspension was kept at room temperature for 6 hours. The Ptcompounds and lipids were then extracted with chloroform (sample 1) orthe sample was lyophilized to eliminate the water and the powderdissolved in methanol (sample 2) (see FIG. 6). Samples 4, 5, and 6 wereprepared by complete evaporation of solvents after keeping the samplesat 40° C. for 6 h, and redissolving them in methanol. Samples 7 and 8were prepared by lyophilization of water for 1-2 d and redissolution inmethanol. All samples were prechecked by HPLC before tracking with NMR.Chemical shifts of the products are expressed in parts-per-millionrelative to Na₂PtCl₆ in ¹⁹⁵Pt and DMPC in ³¹P NMR.

DACH—Pt—Cl₂ Characterization

Yellow precipitates from NMR samples were collected and redissolved inDMF-d₇ to characterize them with ¹H and ¹³C NMR. ¹H NMR (DMF-d₇):1.13-1.17 (m, 2H), 1.46-1.55 (m, 4H), 2.05-2.09 (broad, 2H) 2.55-2.59(m, 2H), and 5.07 and 5.63 (broad s, 2 NH2) ppm. ¹³C NMR (DMF-d7): 24.9(C4, C5), 32.3 (C3, C6), and 64.1 (C1, C2) ppm. ¹⁹⁵Pt NMR (CHCl₃, CH₃OH)1950 ppm, (DMF) 2250 ppm (strong single peak). Elemental analysis: Calc.C(18.99), H(3.69), N(7.38), Pt(51.50); Found C(18.58), H(3.72), N(7.40),Pt(51.30). All these data were confirmed by an authentic sample ofDACH—Pt—Cl₂.

RESULTS Preparation of Liposomal Pt-complexes

NDDP and its two isomers, B10 and L10 were formulated in liposomescomposed of combinations of various lipids including DMPC, DMPG, DOPC,DOPG, PA, PE, and PS. The liposomes were formed by reconstitutingpreliposomal powders containing the Pt-complex and the lipids withunbuffered 0.9% NaCl aqueous solution (saline) or phosphate-bufferedsaline (PBS). The entrapment efficiency (%EE) of all liposomalformulations was >90% and was not significantly affected by the lipidcomposition, reconstitution solution, or NDDP isomer used. No crystalsof free drug were observed in any of these preparations within 24 h asassessed by optic microscopy. The median size of the multilamellarvesicles was 1-2 μm in all preparations. The median size of thesmall-size liposomes prepared by ultrasonication of multilamellarvesicles was 50-100 nm.

Intraliposomal Stability of Pt-complexes 1) Role of the spatialconfiguration of the Pt-complex and pH of the liposome suspension.

FIGS. 2A-D show the stability of liposomal Pt-complex formulations usingsaline or PBS as the reconstitution solution and DMPC:DMPG ratios of 7:3and 3:7. As observed previously [1], the branched configuration of theleaving group of the Pt-complex and the content of DMPG in the lipidbilayers correlated with a higher rate of degradation of the Pt-complex.As a result, the complex with a linear leaving group L10 was highlystable, while the highly branched NDDP was rather unstable, and theminimally branched B10 had an intermediate stability. The use of PBS asthe reconstitution solution resulted in a significantly higher stabilityof the Pt-complexes as compared with saline. For example, 6 h afterliposome preparation, the percentages of intact NDDP in saline versusPBS were 43.7% vs 82.1%, whereas the percentages for B10 were 85.0% vs95.9%, and the percentages for L10 93.1% vs 100%, respectively. The pHof the liposome suspension in saline decreased from 7.0 to 3.8-6.2depending on the Pt-complex, whereas PBS held the pH of the solution toaround 6.0-7.0 in all cases. These results indicate that: 1) an acidicpH enhances the intraliposomal degradation of the Pt-complexes, and 2) agood neutral buffer system can reduce or stop the intraliposomaldegradation of the Pt-complexes. To confirm these results, we tested theintraliposomal stability of the Pt-complexes in strongly acidic (pH=3.0)or basic (pH=8.0) saline solutions prepared by adding 0.1 N HCl or NaOHaqueous solution to pH 7.0 saline. The pH 3.0 saline increased thedegradation rate of all Pt-complexes, whereas the pH 8.0 saline did notinduce any significant Pt-complex degradation even at 24 h afterliposome preparation. All formulations using a relative higher amount ofDMPG (DMPC:DMPG=3:7) displayed a higher rate of Pt-complex degradationin good correlation with the pH of the liposome suspension, because DMPGis an acidic phospholipid.

2) Role of Temperature

The intraliposomal stability of the Pt-complexes was checked at 40° C.and compared with results obtained at room temperature. Pt-complexdegradation was temperature-dependent, with the degradation rates beingabout 30-70% higher at 40° C. than at 25° C., depending on thePt-complex tested.

3) Role of Lipid Composition

Liposomal formulations of NDDP using DMPC:PA, PS:DMPG, and DMPC:PE at a7:3 molar ratio, and DOPC:DOPG at 1:0, 7:3, 3:7, and 0:1 molar ratioswere prepared and tested using saline as the reconstitution solution(FIG. 3). The acidity of phospholipids (PA>PG>PS) and the relative DOPGcontent (DOPC:DOPG 0:1>3:7>7:3>1:0) enhanced the intraliposomaldegradation of NDDP and a good correlation between Pt-complexdegradation and acidic pH was again observed.

The same conclusion was drawn in studies with NDDP in liposomes with thesame DMPC:DMPG molar ratio (7:3) but different NDDP:total lipid ratios(1:5, 1:10, 1:15, and 1:30) and, therefore, different DMPG contents.Using a 1:5 or 1:10 ratio, 85% of initial NDDP was present at 24 hours;in contrast, using a 1:15 or 1:30 ratio resulted in an enhanced NDDPdegradation, with only 25% of the original NDDP remaining at 24 h, thussuggesting a correlation between extent of degradation and absoluteamount of DMPG within the lipid bilayers.

4) Role of Liposome Size

Liposome size did not affect the intraliposomal stability of NDDP:ultrasonication of the original suspension of multilamellar vesicles didnot significantly change the stability of NDDP regardless of the lipidcomposition used.

Correlation Between In Vitro Cytotoxicity and Intraliposomal Stability

We studied the in vitro cytotoxicity of different liposomal Pt-complexpreparations against A2780 cells with the MTT assay and correlated theresults with the intraliposomal stability of the Pt-complex. Results areshown in FIGS. 2A-D. The IC₅₀ values correlated fairly well with drugstability: the more stable the Pt-complex, the less toxic or higher theIC₅₀. When about 20%, 50%, and 90% of the original Pt-complex remainedat 6 h, the IC₅₀ was approximately 3-5, 7-10, and 20-50 g/ml,respectively. These results indicate that the intraliposomal degradationof the Pt-complex is required to exert its cytotoxic effect and is,therefore, an intraliposomal activation step.

Identification of Active Intermediate(s) of NDDP

No new peaks corresponding to the degradation products are observed bythe HPLC method developed for NDDP, either because they elute with thephospholipids or they do not have UV absorbence. Attempts to separateany new peaks from the lipid peaks have been so far unsuccessful.

To keep track of the reaction cascade of liposomal NDDP, we tried toapply the NMR tracking technique used by other researchers [2, 3, 9-11]to characterize the degraded/activated products of NDDP.

1) Formulations prepared using lipids in chloroform solution (samples 1,2, and 3 of FIG. 6A).

By ¹⁹⁵Pt NMR of sample 1 (saline, chloroform extraction, FIG. 4A) andsample 2 (saline, lyophilization, FIG. 4B), the NDDP peak was detectedat 1750 ppm and a new peak corresponding to DACH—Pt—Cl₂ was detected at1950 ppm. Prolonging the reaction time, lowering the pH, increasing thetemperature, and increasing the amount of DMPG in the liposomes enhancedthe degradation/activation of NDDP, increasing the intensity of the peakat ¹⁹⁵0 ppm. However, by ³¹P NMR, no new peaks were observed exceptthose corresponding to DMPC (2 ppm) and DMPG (3 ppm), indicating thatthe new product shown by ¹⁹⁵Pt NMR is not a DMPG-incorporatingPt-complex.

These spectra results were similar to those from sample 3 (FIG. 6A,t-butanol, lyophilization, FIG. 4C), in which liposomal NDDP was notexposed to saline, thus indicating that the presence of CHCl₃ can alsoact as a donor of chloride to form DACH—Pt—Cl₂. After storing theyellowish NMR samples at 4° C. overnight, yellow crystals slowlyprecipitated. The precipitates were filtered, dried, and the structureof the compound was proved to be DACH—Pt—Cl₂ by ¹H, COSY (FIG. 5A), ¹³C(FIG. 5B), and ¹⁹⁵Pt NMR. A yellow precipitate was also observed 2-3weeks after leaving the original liposomal NDDP suspension at roomtemperature. NDDP in liposomes composed of only DMPC did not give anynew peaks by either ¹⁹⁵Pt or ³¹P NMR, which correlates with itscompletely preserved stability in the absence of DMPG.

2) Formulations prepared using lipids in dry powder (samples 4, 5, 6, 7,and 8).

We performed the same tracking experiment with formulations preparedusing lipids in dry powder instead of chloroform solutions to eliminatethe influence of the presence of residual CHCl₃ on the degradation ofNDDP (FIG. 6). In samples 4 and 5 (solvent t-Butanol+water and methanol,respectively) containing lipids and NDDP, no reactions occurred, whereassample 6 (chloroform solution of lipids and NDDP) showed the presence ofDACH—Pt—Cl₂ by ¹⁹⁵Pt NMR. The results with these samples, which wereincubated at 40° C. for 6 h, confirm that the presence of chloroform caninduce the degradation of NDDP into DACH—Pt—Cl₂. Sample 7 was preparedby reconstitution of preliposomal NDDP powder in saline of pH 6.5-7.0for 6 h at room temperature. No significant degradation (<5%) wasobserved, whereas when reconstituted in acidic saline of pH 3.0-4.0(sample 8), a 60-95% degradation of NDDP occurred in 10 min yieldingDACH—Pt—Cl₂ as determined by ¹⁹⁵Pt NMR and HPLC (Table 1).

TABLE 1 Effect of various pH values of saline on intraliposomalstability of NDDP prepared with lipids as dry powder. NDDP stability (%)Time after reconstitution pH of saline 10 min 1 h 2 h 6 h 3.0 43 — 15 75.0 80 72 67 38 7.0 100 — 95 92

Relationship Between Pt-complex Stability and In Vivo AntitumorActivity 1. Preparations Containing Residual Chloroform

These studies were done with liposomal Pt-complex suspensions preparedusing lipids dissolved in chloroform. In in vivo antitumor activitystudies against L1210 leukemia, an inverse relationship between drugstability and antitumor potency was observed (Table 2). The DMPC:DMPGratio was 7:3 for all preparations. The starting pH value of the salineand PBS was 7.0. The tumors were inoculated i.p. on day 0, followed bydrug injection i.p. on day 1.

TABLE 2 In vivo antitumor activity of liposomal Pt complexes against L1210 leukemia. % T/C Dose of Pt complex Reconstitution solution Ptcomplex (mg/kg) Saline PBS NDDP 25 157 114 50 211 171 100 Toxic 200 150Toxic 150 B10 25 114 114 50 128 114 100 228 142 150 163 163 L10 25 100114 50 157 100 100 178 100 150 123 100 Cisplatin 10 142

The optimal doses of liposomal NDDP were 50 mg/kg in saline and 100mg/kg in PBS (%T/C=211 and 200, respectively). For liposomal B10 andL10, the optimal dose was 100 mg in saline (%T/C=228 and 178), but nosignificant antitumor activity was obeserved when both drugs werereconstituted in PBS. In conclusion, the most potent liposomalPt-complex preparations are those with the lowest stability of thePt-complex. However, all formulations had similar antitumor activitywhen administered at the optimal dose.

2. Preparations Not Containing Residual Chloroform

These studies were done with liposomal Pt-complex suspensions preparedwith dry. The relationship between NDDP stability and antitumor activitywas again studies using the in vivo L1210 leukemia model. Table 3 showsthe results with formulations reconstituted with saline solutions ofdifferent pH and administered at different time points afterreconstitutedion. (Values are the means of two separate experiments.)

TABLE 3 Antitumor activity of liposomal NDDP against L 1210 leukemia.Dose of Pt % T/C pH of complex Time of drug administration Drug saline(mg/kg) 10 mm 2 h 6 h NDDP 3.0 12.5 157 25 150 186 240 50 Toxic NDDP 5.012.5 186 25 200 50 133 216 270 100 Toxic NDDP 7.0 25 186 50 200 100 163216 257 Cisplatin 10 150

The optimal doses of liposomal NDDP reconstituted with salines of pH3.0, 5.0, and 7.0 were 25, 50, and 100 mg/kg, respectively. Therefore,the lower the pH, the higher the potency of the preparation in goodcorrelation with the increased intraliposomal drugdegradation/activation. At the optimal doses, the %T/C obtained were214, 271, and 271, respectively. Delaying the time of drugadministration increased the antitumor activity of the formulations, ingood correlation with the increased drug activation with time. However,antitumor activity did not correlate perfectly with the calculatedamount of activated Pt-species formed intraliposomally during theactivation process. For example, 100 mg/kg at pH 7.0 gave a similar %T/Cvalue as 50 mg/kg at pH 5.0, although only 10% of NDDP at pH 7.0 (10mg/kg) and about 70% at pH 5.0 (35 mg/kg) are transformed into theactive Pt-species under those conditions. Further in vivo activationmust, therefore, occur to explain these discrepancies.

DISCUSSION

Our results indicate that NDDP and its isomers are prodrugs ofDACH—Pt—Cl₂ when entrapped in liposomes containing acidic phospholipidsand in the presence of sodium chloride or residual chloroform as donorsof chloride. The rate of transformation of NDDP into DACH—Pt—Cl₂ isdirectly related to the pH of the liposome suspension. The studiesperformed suggest that DMPG and other acidic phospholipids enhance thereaction by providing an acidic milieu within the liposome membranes. Noevidence could be generated to support a direct reaction between NDDPand DMPG to form a DACH—Pt—DMPG complex as one of the activeintermediates of NDDP, as we had previously hypothesized, nor theformation of DACH—Pt aquated species.

DACH—Pt—Cl₂ is the leading compound of the DACH family of Pt-complexes.However, it was never developed because of a lack of solubility inwater. We initially considered this compound for liposome entrapment butdetermined it to be an inappropriate drug for liposome formulationbecause it is insoluble in most organic solvents. DACH—Pt—Cl₂ has only agood solubility in dimethylformamide (DMF), which has a very highboiling point and, therefore, can not be used to prepare liposomes usingthe standard evaporation methods and it is not soluble in any of theorganic solvents used for the lyophilization methods. Our studiesindicate that DACH—Pt—Cl₂ can be generated within the liposome membranesunder the conditions described and that the drug remains liposome-boundwithout leaking out and crystallizing for at least 24 h. In contrast,DACH—Pt—Cl₂ precipitates quickly when formed from NDDP by the additionof HCl. These results constitute the first example of a liposomeformulation in which the compound is synthesized in situ from anentrapped precursor and the liposomes prevent its spontaneousprecipitation. The results are encouraging because they may suggest anavenue for the development of a much needed delivery system for thisvery interesting compound.

A potential strategy is to use a two-step reconstitution procedure bywhich an acidic saline solution is used first to induce the fasttransformation of >80% of NDDP into DACH—Pt—Cl₂, followed after apredetermined period of time by the addition of a buffer solution tobring the pH to >7.0 and to stop the reaction.

Liposomes in accordance with the present invention can be prepared fromvarious amphipathic substances including natural or syntheticphospholipids. Numerous suitable phospholipids are well known in theart. The liposomes of the present invention can be multilamellar,unilamellar, or have an undefined lamellar construction. Apharmaceutical composition comprising such liposomes can include apharmaceutically acceptable carrier or diluent, as well as otherpharmaceutically acceptable adjuvants.

Liposome compositions of the present invention can be used to inhibitthe growth of tumor cells in mammals, particularly in humans. Thecompositions of the present invention should be useful for treatment ofvarious human malignancies, in particular any platinum-sensitive cancer,including ovarian, testicular, lung, head and neck, esophageal, andbladder tumors, sarcomas, lymphomas, and mesotheliomas. Methods of usingthe compositions of the present invention involve administering to amammal an amount of the compositions effective to inhibit tumor growth.The administering step can suitably be parenteral and by intravenous,intraarterial, intramuscular, intralymphatic, intraperitoneal,subcutaneous, intrapleural, or intrathecal injection, or by topicalapplication or oral dosage. Such administration is preferably repeatedon a timed schedule until tumor regression or disappearance has beenachieved, and may be used in conjunction with other forms of tumortherapy such as surgery or chemotherapy with different agents. The doseadministered of a composition in accordance with the present inventionis preferably between approximately 100 and 750 mg/kg of body weight ofthe mammalian subject to which it is administered.

The preceding description of specific embodiments of the presentinvention is not intended to be a complete list of every possibleembodiment of the invention. Persons skilled in this field willrecognize that modifications can be made to the specific embodimentsdescribed here that would be within the scope of the present invention.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

1. Han, I.; ling, Y.-H.; Al-Baker, S.; Khokhar, A. R.; Perez-Soler, R.Cellular pharmacology of liposomalcis-bis-neodecanoato-trans-R,R-1,2-diaminocyclohexane-platinum(II) inA2780/S and A2780/PDD cells. Cancer Res. 1993, 53, 4913-4919.

2. Hollis, L. S.; Miller, A. V.; Amundsen, A. R.; Schurig, J. E.; Stem,E. W. cis-Diamineplatinum(II) complexes containing phosphono carboxylateligands as antitumor agents. J. Med. Chem. 1990, 33, 105-111.

3. Ismail, I. M.; Sadler, P. J. ¹⁹⁵Pt- and ¹⁵N-NMR studies of antitumorcomplexes. In: Platinum, gold, and other metal chemotherapeutic agents;Lippard, S. J. (eds.) American Chemical Society. 1983; pp 171-189.

4. Khokhar, A. R.; Al-Baker, S.; Brown, T.; Perez-Soler, R. Chemical andbiological studies on a series of lipid-soluble (trans-(R,R)- and-(S,S)-1,2-diaminocyclohexane)platinum(II). J. Med. Chem.1991,34,325-329.

5. Perez-Soler, R.; Yang, L. Y.; Drewinko, B.; Lauterzstain, J.;Khokhar, A. R. Increased cytotoxicity and reversal of resistance ofcis-diamminedichloro platinum(II) with entrapment ofcis-bis-neodecanoato-trans-R,R-1,2-diaminocyclohexaneplatinum(II) inmultilamellar lipid vesicles. Cancer Res. 1988, 48, 4509-4512.

6. Perez-Soler, R.; Khokhar, A. R.; Lautersztain, J.; Al-Baker, S.;Francis, K.; Macias-Kiger, D.; Lopez-Berestein, B. Clinical developmentof liposomal platinum. J. Liposome Res. 1990, 1, 437-449.

7. Perez-Soler, R.; Khokhar, A. R. Lipophilic cisplatin analoguesentrapped in liposomes: role of intraliposomal drug activation inbiological activity. Cancer Res. 1992, 52, 6341-6347.

8. Perez-Soler, R.; Han, I.; Al-Baker, S.; Khokhar, A. R. Lipophilicplatinum complexes entrapped in liposomes: improved stability andpreserved antitumor activity with complexes containing linear alkylcarboxylato leaving groups.

Cancer Chemother. Pharmacol. 1994,33,378-384.

9. Qu, Y.; Farrell, N. J. Effect of diamine linker on the chemistry ofbis(platinum) complexes. A comparison of the aqueous solution behaviorof 1,4-butanediamine and 2,5-dimethyl-2,5-hexanediamine complexes. J.Inorg. Biochem. 1990, 40, 255-264.

10. Qu, Y.; Farrell, N. J. Interaction of bis(platinum) complexes withthe mononucleotide 5′-guanosine monophosphate. Effect of diamine linkerand the nature of the bis(platinum) complex on product formation. J. Am.Chem. Soc. 1991, 113, 4851-4857.

11. Slavin, L. L.; Bose, R. N. Phosphonato complexes of platinum(II):kinetics of formation and phosphorus-31 NMR characterization studies. J.Inorg. Biochem. 1990, 40, 339-347.

We claim:
 1. A liposomal antitumor composition, comprising a platinumcomplex having the formula R₁—Pt —X₂ entrapped in a liposome, where theliposome comprises dioleyl phosphatidyl glycerol, and where R₁ isdiaminocycloalkyl and X is halogen.
 2. The composition of claim 1, whereR₁ has from 3 to 6 carbon atoms.
 3. The composition of claim 1, where R₁is 1,2-diaminocyclohexane.
 4. The composition of any one of claims 1-3,where X is chlorine.
 5. The composition of claim 1, where the liposomefurther comprises dioleyl phosphatidyl choline.
 6. The composition ofclaim 1, where the platinum complex is intercalated between the bilayersof the liposome.
 7. A liposomal antitumor composition, comprising aplatinum complex having the formula DACH—Pt—Cl₂ intercalated between thebilayers of a liposome, where the liposome comprises dioleylphosphatidyl glycerol, and where DACH is diaminocyclohexane.
 8. A methodof inhibiting tumor growth, the method comprising administering to amammal a composition, in an amount effective to inhibit tumor growth,the composition comprising a platinum complex having the formulaR₁—Pt—X₂ entrapped in a liposome, where the liposome comprises dioleylphosphatidyl glycerol, and where R₁ is diaminocycloalkyl and X ishalogen.
 9. The method of claim 8, where R₁ has from 3 to 6 carbonatoms.
 10. The method of claim 8, where R₁ is 1,2-diaminocyclohexane.11. The method of any one of claims 8-10, where X is chlorine.
 12. Themethod of claim 8, where the liposome further comprises dioleylphosphatidyl choline.
 13. The method of claim 8, where the complex isintercalated between bilayers of the liposome.
 14. A method ofinhibiting tumor growth, the method comprising administering to a mammala composition, in an amount effective to inhibit tumor growth, thecomposition comprising a platinum complex having the formula DACH—Pt—Cl2intercalated between the bilayers of a liposome, where the liposomecomprises dioleyl phosphatidyl glycerol, and where DACH isdiaminocyclohexane.
 15. A liposomal antitumor composition comprising aplatinum complex, the composition formed by a method, the methodcomprising adjusting the pH of a composition containing aliposome-entrapped first compound, so that the pH is made acidic, saidfirst compound having the formula

where R₁ is diaminocycloalkyl and R₂ and R₃ independently have theformula

where R₄, R₅ and R₆ are each independently hydrocarbon moieties havingfrom 1 to about 10 carbon atoms, and where said liposome comprisesdioleyl phosphatidyl glycerol.
 16. The liposomal antitumor compositionof claim 15, wherein said adjusting comprises exposing theliposome-entrapped, first compound to a solution having an acidic pH.17. The liposomal antitumor composition of claim 15 wherein the methodfurther comprises before said adjusting step, the step of entrappingsaid first compound in said liposome.
 18. The liposomal antitumorcomposition of claim 15 wherein said adjusting comprises reconstitutinga lyophilized composition containing the liposome-entrapped firstcompound using an acidic solution.
 19. The liposomal antitumorcomposition of claim 15 wherein said pH made acidic is between 2 and6.5.
 20. The liposomal antitumor composition of claim 15 wherein saidadjusting comprises adding an acidic solution.
 21. The liposomalantitumor composition of claim 20 wherein said acidic solution comprisessodium chloride.
 22. The liposomal antitumor composition of claim 21wherein said acidic solution is an aqueous solution.
 23. A liposomalantitumor composition comprising a platinum complex, the compositionformed by a method, the method comprising adjusting the pH of acomposition containing a liposome-entrapped first compound, so that thepH is made acidic, said first compound having the formula

where R₁ is diaminocycloalkyl and R₂ and R₃ independently have theformula

where R₄, R₅ and R₆ are each independently hydrocarbon moieties havingfrom 1 to about 10 carbon atoms, said platinum complex having theformula R₁—P_(t)—X₂ where R₁ is as defined above and X is halogen, andwhere said liposome comprises dioleyl phosphatidyl glycerol.
 24. Aliposomal antitumor composition comprising a platinum complex, thecomposition formed by a method, the method comprising adjusting the pHof a composition containing a liposome-entrapped first compound in thepresence of sodium chloride, so that the pH is made acidic, said firstcompound having the formula

where DACH is 1 ,2-diaminocyclohexane and R₂ and R₃ independently havethe formula

where R₄, R₅ and R₆ are each independently hydrocarbon moieties havingfrom 1 to about 10 carbon atoms, said platinum complex having theformula DACH—P_(t) —Cl₂ where DACH is as defined above, and where saidliposome comprises dioleyl phosphatidyl glycerol.
 25. The liposomalantitumor composition of claim 23 wherein said adjusting comprisesadding an acidic solution.
 26. The liposomal antitumor composition ofclaim 24 wherein said adjusting comprises adding an acidic solution. 27.A liposomal antitumor composition comprising a platinum complex, thecomposition formed by a method, the method comprising entrapping a firstcompound in a liposome comprising an acidic phospholipid that is dioleylphosphatidyl glycerol to produce a liposomal composition, said firstcompound having the formula

where R₁ is diaminocycloalkyl and R₂ and R₃ independently have theformula

where R₄, R₅ and R₆ are each independently hydrocarbon moieties havingfrom 1 to about 10 carbon atoms.
 28. The liposomal antitumor compositionof claim 27, wherein the method further comprises the steps oflyophilizing said liposomal composition to produce a lyophilizedliposomal composition and reconstituting said lyophilized liposomalcomposition in an aqueous solution.
 29. The liposomal antitumorcomposition of claim 27, wherein the method further comprises the stepsof lyophilizing said liposomal composition to produce a lyophilizedliposomal composition and reconstituting said lyophilized liposomalcomposition in a solution comprising sodium chloride.
 30. The liposomalantitumor composition of claim 27, wherein the method further comprisesthe steps of lyophilizing said liposomal composition to produce alyophilized liposomal composition and reconstituting said lyophilizedliposomal composition in an acidic solution.
 31. A liposomal antitumorcomposition comprising a platinum complex, the composition formed by amethod, the method comprising entrapping a first compound in a liposomecomprising an acidic phospholipid that is dioleyl phosphatidyl glycerolto produce a liposomal composition, said first compound having theformula

where R₁ is diaminocycloalkyl and R₂ and R₃ independently have theformula

where R₄, R₅ and R₆ are each independently hydrocarbon moieties havingfrom 1 to about 10 carbon atoms, said platinum complex having theformula R₁—P_(t)—X₂ where R₁ is as defined above and X is halogen.
 32. Aliposomal antitumor composition comprising a platinum complex, thecomposition formed by a method, the method comprising entrapping a firstcompound in a liposome comprising an acidic phospholipid that is dioleylphosphatidyl glycerol to produce a liposomal composition, said firstcompound having the formula

where DACH is 1 ,2-diaminocyclohexane and R₂ and R₃ independently havethe formula

where R₄, R₅ and R₆ are each independently hydrocarbon moieties havingfrom 1 to about 10 carbon atoms, said platinum complex having theformula DACH—P_(t)—Cl₂ where DACH is as defined above.
 33. The liposomalantitumor composition of claim 31 or 32 wherein the method furthercomprises the steps of lyophilizing said liposomal composition toproduce a lyophilized liposomal composition and reconstituting saidlyophilized liposomal composition in an aqueous solution.
 34. Theliposomal antitumor composition of claim 31 or 32 wherein the methodfurther comprises the steps of lyophilizing said liposomal compositionto produce a lyophilized liposomal composition and reconstituting saidlyophilized liposomal composition in an acidic solution.
 35. The methodof claim 8, wherein said tumor is ovarian cancer, testicular cancer,lung cancer, cancer of the head or neck, esophageal cancer, bladdercancer, a sarcoma, a lymphoma or a mesothelioma.
 36. The method of claim14, wherein said tumor is ovarian cancer, testicular cancer, lungcancer, cancer of the head or neck, esophageal cancer, bladder cancer, asarcoma, a lymphoma or a mesothelioma.
 37. A method for treating cancer,the method comprising administering to a mammal in need thereof thecomposition of any one of claims 7, 15, 23, 24, 27, 31, or
 32. 38. Themethod of claim 37, wherein said mammal is a human.
 39. The method ofclaim 37, wherein said mammal has a cancer that is ovarian cancer,testicular cancer, lung cancer, cancer of the head or neck, esophagealcancer, bladder cancer, a sarcoma, a lymphoma or a mesothelioma.
 40. Apharmaceutical composition comprising an amount of the composition ofclaim 1 effective to treat cancer and a pharmaceutically acceptablecarrier or diluent.
 41. A pharmaceutical composition comprising anamount of the composition of claim 7 effective to treat cancer and apharmaceutically acceptable carrier or diluent.
 42. A pharmaceuticalcomposition comprising an amount of the composition of any one of claims15, 23, 24, 27, 31 or 32 effective to treat cancer and apharmaceutically acceptable carrier or diluent.
 43. The liposomalantitumor composition of claim 15 wherein said adjusting is in thepresence of sodium chloride.
 44. A method for treating cancer, themethod comprising administering to a mammal in need thereof thecomposition of any one of claims 16—22, 25, 26, 28, 20, 32.